Improving Aptamer Affinity and Determining Sequence-Activity Relationships via Motif-SELEX

The affinity of nucleic acid aptamers isolated in vitro via Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is often limited because all of the potential nucleic acid sequence space cannot be screened. In this study, we introduce motif-SELEX, a novel method that enables the optimization of existing aptamers by generating libraries that broadly represent both sequence and length variations of the parent sequence. This approach enables the isolation of sequences with improved affinity without the biases and limitations of traditional mutagenesis methods like doped SELEX and error-prone PCR. As a demonstration, we applied Motif-SELEX to a DNA-based morphine aptamer and an RNA-based apixaban aptamer, achieving 3-10-fold improvements in affinity and discovering new sequences with variable domain lengths that would be inaccessible to traditional SELEX methods. Critically, our analysis of motif-SELEX pools also enabled us to identify sequence and structural elements crucial for target binding, and to predict secondary and tertiary structures for a given aptamer family - even when those structures involve non-canonical nucleotide interactions. We believe that motif-SELEX offers a versatile generalizable solution for optimizing the structure and binding properties of functional nucleic acid molecules for diverse applications.